1. Field of the Invention
The field of the invention is improved dry reagents for instrumented whole blood tests useful for diabetics.
2. Description of the Related Art
Blood glucose monitoring has revolutionized the treatment of diabetes. Large-scale clinical trials have demonstrated that frequent blood glucose monitoring can aid in the prevention of many of the long-term complications of diabetes, such as diabetic retinopathy, circulatory disorders, and death. After nearly twenty years of development, blood glucose monitoring has now become a several billion dollar a year business.
As the blood glucose-monitoring field has advanced, the various blood glucose monitors have become more and more generic. All possess good accuracy, ease of use, and speed. As a result, the various manufacturers of blood glucose monitors have focused major efforts on gaining minor technical advantages to make minor improvements in their respective market shares. Such improvements may include minor improvements in speed, blood sample size, ease of sample application, cost, etc. All, however, produce test strips that measure only blood glucose.
Although blood glucose is the most important biochemical parameter to measure in diabetes, it is not the only parameter of medical interest. Other parameters of medical relevance include glycosylated hemoglobin, used to measure long-term blood glucose control, ketone levels, used to indicate if the patient is at risk for diabetic ketoacidosis, and lipids such as cholesterol, triglycerides, lipoproteins, and chylomicrons, used to indicate the patient's relative risk of cardiovascular disease.
In this document, ketoacidosis and elevated triglycerides will be used as the main examples of other biochemical parameters that are medically relevant to the treatment of diabetes, however it should be understood that the methods discussed here are general purpose, and may be used for a wide variety of different analytes.
Diabetic ketoacidosis is a major complication of diabetes. Such conditions occur during times of extreme insulin deficiency. Here the diabetic's tissues are unable to process glucose, and as a result, initiate the biochemical processes that result in the formation of ketones and excess blood glucose. During periods of insulin starvation, body cells are unable to metabolize glucose as an energy source and instead metabolize fat as an energy source. Ketone bodies, made up of acectoacetate, acetone, and beta-hydroxybutyrate (also called D-3-hydoxybutyrate) are produced from this fat metabolism process, and these build up in the blood. Excessive levels of ketone bodies in turn can alter the pH balance of the blood to a more acidic state, as well as other undesirable complications, eventually leading to confusion, coma, and death. In the early stages of fat metabolism, the ketone bodies contain relatively large amounts of acectoacetate and acetone. However in more profound ketoacidosis, the ketone bodies contain primarily beta-hydroxybutyrate.
Each year, about 12 out of every 1000 diabetics are hospitalized for Ketoacidosis, and 2% of those hospitalized die from it. It is the commonest cause of death for diabetic children.
Early detection is the best way to prevent diabetic ketoacidosis. If detected in time, rehydration and low-dose insulin therapy can be used to treat ketoacidosis. Thus means to ensure that the onset of ketoacidosis is promptly detected are of extreme utility to diabetics.
Although ketoacidosis is a major problem, the biggest complication of diabetes is cardiovascular disease. Two out of three diabetics ultimately die from heart disease and stroke (caused by cardiovascular disease), and many others suffer from other cardiovascular disease complications such as diabetic retinopathy. Much of this cardiovascular disease in turn is caused by the build-up of fatty deposits (lipid rich plaque) in blood vessels and arteries.
Diabetics, and in particular type 2 diabetics, often have an abnormally large increase in the amount of triglycerides, lipids, and lipoproteins circulating in the blood after meals. This increase is particularly severe for type 2 diabetics who have just eaten meals with a high fat content. This post-meal lipoprotein increase is often referred to as “postprandial lipemia” In postprandial lipemia, a large number of triglyceride-rich chylomicrons, low-density lipoproteins (LDL), very low-density lipoproteins (VLDL) and other lipoproteins are released from the small intestine. These triglyceride-rich chylomicrons and other lipoproteins scatter light, and often cause the plasma and serum from postprandial subjects to have so much optical turbidity that this turbidity interferes with the optical determination of other analytes. As a result, for many clinical analytes, it is a routine clinical practice to require patients to fast for at least twelve hours before providing blood samples.
Recent studies have shown that this postprandial lipemia can do more harm than just generate turbid plasma. The LDL and chylomicron lipoprotein particles tend to build up on the walls of arteries, leading to atherosclerosis (fat deposits on artery walls) and subsequent increased risk of coronary artery disease, stroke, and other cardiovascular disorders.
Fortunately the choice between a high-fat diet that causes substantial postprandial lipemia, and a low-fat diet that avoids high postprandial lipemia, is a relatively easy choice to implement—substitute low-fat foods for high-fat foods. If type 2 diabetics, who are at a particularly high risk for atherosclerosis and other cardiovascular complications caused by postprandial lipemia, and who are accustomed to routinely testing postprandial blood glucose levels, also had a simple way of determining their relative level of postprandial lipemia at the same time, they would be constantly reinforced to chose low-fat diets, and thus could substantially reduce their risk of cardiovascular disease.
Returning to the ketoacidosis example, means to measure ketone levels are known in the art. These include visually read test strips for acetone or acectoacetate in the urine, as well as whole blood tests for beta-hydroxybutyrate. Diabetics are trained that whenever their glucose levels are high, they should follow up by immediately running a separate ketone test.
Examples of urine ketone dry reagent tests include Ketostix, Keto-Diastix (Beyer) or Chemstrip K (Roche). Such urinary tests generally use non-enzymatic detection methods (such as nitroprusside based chemistries) that are primarily sensitive to acectoacetate, slightly sensitive to acetone, and not at all sensitive to beta-hydroxybutyrate. One drawback of tests that measure only urinary acectoacetate or acetone is that such tests can miss or underreport extreme levels of ketoacidosis. In mild ketosis, the body produces acectoacetate, acetone and beta-hydroxybutyrate in relatively proportionate amounts, and thus urinary tests for acectoacetate and acetone will detect mild ketosis. However in extreme ketoacidosis, the body produces mostly beta-hydroxybutyrate and relatively small amounts of acectoacetate and acetone. Thus non-enzymatic nitroprusside based acectoacetate and acetone sensitive tests may become insensitive to extreme ketoacidosis right when they are needed the most.
Simple dry reagent whole blood tests for beta-hydroxybutyrate, the most clinically relevant indicator of ketoacidosis, are known in the art. Presently, such dry reagent tests use a disposable reagent that performs only the beta-hydroxybutyrate test. Often this disposable beta-hydroxybutyrate reagent is read in a meter that is capable of reading a number of different types of single test reagents. For example, GDS diagnostics, Elkhart Ind., sells the “Stat-Site™” meter, which can read separate calorimetric dry reagent tests for either whole blood glucose or ketones (beta-hydroxybutyrate). This technology is taught in U.S. Pat. No. 5,139,685. Polymer Technology Systems of Indianapolis Ind. sells the Bioscanner™ meter that can also read separate calorimetric dry reagent tests for either whole blood glucose or ketones. Similarly, MediSense sells the “Precision Xtra™” meter that can read separate electrochemical dry reagent tests for either glucose or beta-hydroxybutyrate.
Other one-meter multiple-reagents systems are in commercial use. The LXN Corporation sells the “Duet™” and “In Charge System™” meters that are capable of reading either a calorimetric glucose dry reagent test, or alternatively a colorimetric glycated protein (fructosamine) dry reagent test. These are discussed in more detail in U.S. Pat. Nos. 5,695,949 and 6,027,692.
Although diabetics are accustomed to testing their blood glucose several times a day, they may often forget to run a ketone test, since such tests require extra reagents and effort. Indeed, in an effort to correct for this normal human lapse, some glucose meters, such as the LifeScan “ultra” blood glucose system, will attempt to remind users to run ketone tests by an extra “Ketones?” meter prompt. However, clearly many diabetics will ignore this reminder.
Returning to the lipemia example, methods to measure postprandial lipemia are also known in the art. These tests include standard enzymatic tests for triglycerides, lipoprotein precipitation tests using chemical agents that selectively precipitate lipoproteins from plasma, and immunoprecipitation tests for specific lipoproteins (using specific anti-lipoprotein antibodies). Studies have also shown that there is a good correlation between the amount (level, concentration) of plasma or serum chylomicrons and the turbidity (light scattering) of the plasma or serum. Tazuma et. al. (“A quantitative assessment of serum chylomicron by light scattering intensity: Application to the intestinal fat absorption test”, Journal of Gastroenterology and Hepatology, Volume 12(11), November 1997, pp 713-718) utilized this correlation to devise a clinical test for serum chylomicrons based on light scattering nephelometric, (turbidimetric) methods. Tazuma et. al. found that a linear relationship existed between serum light scattering (using serum diluted 1:10 into 0.9% saline) and triglyceride concentration. Specifically, in Tazuma's system, this relationship was shown by equation 1 below:y=0.33[x]+14.969x  Equation 1
Here “y” is the serum chylomicron triglyceride concentration (level) in mg/dl, and “x” is the relative extent of plasma light scattering on Tazuma's Nippon Shoji Micronephelometer MN-202, used for this experiment.
Although Tazuma's work shows that it is possible to use light scattering measurements to determine triglyceride levels in diluted serum, this is an unusual approach that has not previously been used for whole blood dry reagent tests. More typically, whole blood triglyceride dry reagent tests are based upon enzymatic reactions that produce a colored reaction product and are measured by a calorimetric instrument. Examples of this type of test include the Polymer Technology Systems (Indianapolis, Ind.) “Cardiocheck” system, and the Polymer Technology Systems “Lipid Panel” test strips. The “Lipid Panel” test strips measure total cholesterol, HDL (high density lipoprotein), and triglycerides using plasma obtained from whole blood by filtering the blood through a spreading layer, a blood separation layer, and a fractionation layer. The resulting purified plasma is then read in three separate enzymatic reaction zones, each zone containing a different enzymatic chemistry that generates a colorimetric reaction.
The Cholestech LDX analyzer (Cholestech corporation, Hayward, Calif.), exemplified by U.S. Pat. Nos. 5,110,724; 5,114,350 and 5,171,688 is another dry reagent triglycerides test that also measures total cholesterol, HDL, and triglycerides by a similar process in which whole blood is first fractionated into plasma, and then read in three separate enzymatically based calorimetric reaction pads. Due to the need to separate whole blood into plasma prior to contact with the various enzymatic reaction zones, both systems require relatively large amounts of blood and both systems are relatively slow The PTS Lipid panel test requires 40 ul of blood and requires two minutes to perform a test. The Cholestech LDX system requires approximately 60 ul of blood and requires about five minutes to perform a test. As a result, neither approach would be competitive in the blood glucose market, where sample sizes are invariably less than 20 ul, and test times are often only a few seconds are less.
Ideally, what is best from a medical perspective is a blood glucose test that automatically (without any extra user thought, process, or intervention) also reports blood beta-hydroxybutyrate levels, or blood lipid (triglyceride or chylomicron) levels, or other important second analyte levels, using the same drop of blood used to perform the standard and habitual glucose test. Indeed such a combined test would save many lives by facilitating the early detection of ketoacidosis, prevention of atherosclerosis, or other complication of diabetes. Additionally, such combined tests would be of strong commercial interest as well, since if everything else were equal, a combined glucose/beta-hydroxybutyrate test, glucose/triglycerides test, glucose/lipoprotein test, glucose/chylomicron test, or glucose/relevant-second-analyte test would be strongly preferred by diabetics over the glucose-only tests presently used.
However no such single-blood-drop-activated, combined blood-glucose/blood-beta-hydroxybutyrate dry reagent or combined glucose/lipoprotein reagent has previously been proposed, invented, or commercialized.
By contrast, combined glucose-ketone test strips have been available for urine testing for many years. Given the competitive nature of the blood glucose-monitoring field, why does this discrepancy exist between the long-term commercialization of combined urine glucose-ketone dry reagent test strips, and the complete lack of any prior art in combined high speed, low blood sample, whole blood glucose/beta-hydroxybutyrate or blood glucose-second analyte dry reagent tests?
The difference is almost certainly due to the radically different nature of the two different sample types. Urine is available in large (100+ milliliter [ml]) quantities. It is nearly transparent. Thus a combined glucose-ketone dry regent test may be made by simply putting a calorimetric glucose dry reagent test pad onto solid support a certain distance away from a colorimetric ketone dry regent test pad. Because large amounts of sample are present, the distance between the two test pads can be so great as to minimize any “cross talk” due to reaction intermediate or colorimetric dye indicator diffusion between the two pads.
It is often the case in nearly every area of technology that devices optimized for a single purpose outperform devices optimized for multiple purposes. Blood glucose testing has been a mature field for nearly twenty years, and blood glucose meters and reagents have evolved to a highly advanced state. Patients and physicians are unlikely to accept a dual glucose-beta-hydroxybutyrate or glucose-lipemia reagent as being a genuine improvement unless, at a minimum, the glucose portion of the reagent performs at a level that is competitive with stand-alone blood glucose tests. If the combined reagent requires no extra user effort, the blood glucose portion is competitive, and the extra cost for the secondary function is minor, then the user will benefit and the combined reagent will likely be a medical and commercial success.
In this context, the commercial success of combined urine-ketone test strips can be understood. These devices function with the same urine sample and require no additional user effort. The urine glucose part of a combined urinary glucose-ketone test strip performs as well as stand-alone urine glucose test strip.
By contrast, combined whole blood glucose-beta-hydroxybutyrate or other relevant glucose-second analyte dry reagents must overcome some formidable technical challenges. Whereas urine samples typically have a volume of 100 ml (milliliters), blood samples, typically derived from a fingerstick, are more typically have a volume around 1-10 ul (microliters), or more generally from about 0-20 ul. This is nearly five orders of magnitude less in size. Whereas urine is nearly transparent and relatively free of optical and electrochemical interfering substances, blood is intensely colored and contains nearly 50% hemoglobin and other strong optical and electrochemical interfering substances.
In order to meet the requirement for no additional user effort, a whole blood combined glucose-ketone/beta-hydroxybutyrate or other relevant glucose-second analyte test must place both the glucose sensing means and the ketone/beta-hydroxybutyrate (or other second analyte sensing means) close enough together as to both be activated with the same small (1-10 ul, or 0-20 ul) drop of whole blood. Further, the test must be designed to minimize “cross talk” between such closely spaced sensing means.